Prenylated flavin mononucleotide
# Prenylated flavin mononucleotide

Prenylated flavin mononucleotide (prFMN) is a cofactor biosynthesized by the flavin prenyltransferase UbiX and used by UbiD enzymes for reversible decarboxylation reactions. Hence, prFMN is pivotal for catalysis in the ubiquitous microbial UbiD/X system.

prFMN is flavin prenylated at the N5 and C6 positions resulting in the formation of a fourth non-aromatic ring.

prFMN was discovered in 2015 at the University of Manchester by David Leys' group.

    Structure of FMN and $prFMN^{iminium}.$ The leftmost structure shows FMN (Flavin mononucleotide), the rightmost structure shows $prFMN^{iminium}$ (prenylated flavin mononucleotide), Note the presence of the quaternary carbon (C3’) in $prFMN^{iminium}$ indicative of cofactor prenylation.

Two studies in 2015 characterized UbiX as a flavin prenyltransferase, supplying prFMN to UbiD/Fdc1 which utilises the cofactor to catalyse a reversible decarboxylation reaction. Ferulic acid decarboxylase (Fdc1) from A. niger co-expressed in E.coli with UbiX from E.coli $(AnFdc1^{UbiX})$ once purified had clear spectral differences to singly expressed AnFdc1, and was capable of in vitro decarboxylation of a range of aromatic carboxylic acids. The atomic resolution of the crystal structure of $AnFdc1^{UbiX},$ allowed elucidation of the structure of the modified FMN cofactor classified as prFMN. The crystal structure revealed an isopentenyl-adduct to the N5-C6 of FMN, with the modifications branched nature and the position of the covalent linkages with flavin suggesting prenylation.

    $prFMN^{iminium}$ in the active site of AnFDC1. PDB file: 4ZA4.

## $PrFMN^{Ox}$

UbiD activation by UbiX/prFMN was found to be dependent on oxygen suggesting that the reduced prFMN product of UbiX is oxidised to the catalytically relevant form. Several variations of the oxidised prFMN $(prFMN^{ox})$ cofactor were observed: $prFMN^{iminium},$ hydroxylated $prFMN^{iminium}$ and $prFMN^{ketimine}.$ Determination of the prFMN isomer that was catalytically relevant involved incubation of $AnFdc1^{UbiX}$ with phenylpyruvate (of which a small proportion is α-hydroxycinnamic acid which closely resembles cinnamic acid - a model substrate). Incubation with phenylpyruvate lead to an altered UV-Vis spectrum and reversible enzyme inhibition. The crystal structure of $AnFdc1^{UbiX}$ with phenylpyruvate revealed a bond between C1’ of $prFMN^{iminium}$ and a phenylacetaldehyde adduct – a species that can be formed by decarboxylation of α-hydroxycinnamic acid and tautomerization of the α-hydroxystyrene $prFMN^{iminium}$ adduct.

This observation confirmed that it is the $prFMN^{iminium}$ that is the catalytically relevant cofactor.

